The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
Molecular microscopy is a non-invasive electron imaging technology that uses advanced specimen preparation and imaging methods designed specifically to visualize complex biological samples, under conditions close to their native state. Samples are preserved in a very thin film of solution on an EM grid, by vitrification (using an automated cryogenic robot) or by negative stain, and then imaged using a transmission electron microscope (TEM) controlled by automated software that enables sampling of a significant portion of the specimen. High-throughput molecular microscopy combines robotic instruments, automated data collection and processing software, and a relational database into a pipeline to prepare, image, and analyze samples in a reproducible manner and with throughputs capable of addressing biopharmaceutical characterization needs in a statistically significant manner. For well-ordered samples such as viruses, and virus-antibody complexes, the achievable resolution can be <0.4 nm.
In preparing EM grids for molecular microscopy, preparation of the sample grid is often the least controlled step in the process. Typically, a few microliters of sample are applied to a specimen grid. Excess sample is removed by contact blotting with a piece of absorbent paper to thin the film of solution. Finally, vitrification, achieved by plunging the thin sample into a liquid coolant (typically liquid ethane). The ultimate thickness of the sample is related to, among other things, the relative hydrophilicity of the grid surface, the relative humidity and temperature of the environment, and the length of blotting time. In addition, once the solution is applied to the grid it has an extremely high surface to volume ratio and its behavior is dominated by the heat and mass transfer that result from evaporation of the aqueous sample. The result of combining these events is that grid surfaces are not reproducible, either within the same grid or within grids. Portions of the specimen grid will be too thick for effective penetration by the electron beam, while others will be too thin for proper specimen preservation, while still others will suffer from increases in solute concentrations due to evaporation of water.
Previously described vitrification robots comprise a guillotine-like grid plunger. The vertical motion is provided by a driven rod to which standard forceps clamping an EM grid are attached. The blotting action is provided by blotting paper attached to rotating disks that are arranged to close on the sample like “clapping hands.” The disks rotate to provide a fresh blotting surface for each clap and the duration and pressure of each action are precisely controlled. The entire apparatus is enclosed in an environmental chamber. A shutter at the bottom of the chamber provides access to the rod to load new forceps/grids and to plunge the sample into the coolant. The operational sequence begins with lowering the rod under stepper motor control and attaching the forceps and grid through the opened shutter. The rod retracts and the shutter closes. The sample solution is loaded onto the grid through a hole in the chamber using a pipette to extract solution from a vial inside the chamber, and deposit a controlled amount (typically 3 μL) on the grid. The grid is positioned for blotting and a controlled sequence of blotting actions is performed. The grid is allowed to drain/thin for a controlled period. Finally, the rod is disengaged from the stepper motor and pneumatically propelled (approx. 2 m/sec) through the shutter into the coolant. After vitrification, the coolant reservoir engages the rod/forceps/sample assembly and can be removed from the system to permit further manipulation and transfer to the TEM using conventional cryotransfer tools. All aspects of the preparation sequence are controlled by a computer system that includes an easy-to-use operator interface. See, e.g., Iancu et al., Nat. Protocols 1: 2813-2819, 2007.
Although improving the sample preparation through automating the steps into a more reproducible form, previously described vitrification robots still do not provide the level of sample control necessary for truly reproducible molecular microscopy.